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Increased Cholesterol Efflux Capacity is Associated with Improved Survival in Heart Transplant Recipients
The 18th Annual Scientific Meeting
HFSA
S81
Molecular Biology and Genetics II 204 Mechanism of Fibrosis in Inflammatory Dilated Cardiomyopathy Daiva Bironaite1, Dainius Daunoravicius2, Julius Bogomolovas3, Sigitas Cibiras2, Dalius Vitkus2, Edvardas Zurauskas2, Ieva Zasytyte2, Siegfried Labeit3, Algirdas Venalis1, Virginija Grabauskiene2; 1State Research Institute, Center for Innovative Medicine, Vilnius, Lithuania; 2Vilnius University, Faculty of Medicine, Vilnius, Lithuania; 3Universit€atsmedizin Mannheim, Mannheim, Germany Background: Inflammation plays a significant role in myocardial damage and is considered to be involved in the pathogenesis of myocarditis, which often progresses to inflammatory dilated cardiomyopathy (iDCM). Myocarditis is known to result in fibrosis, inflammatory exudates, adverse ventricular remodeling and, eventually, cardiac failure. The level of fibrosis in iDCM is supposed to vary depending on inflammatory intensity and duration. Objectives: The purpose of our study was to investigate the molecular mechanisms regulating fibrogenesis and its role in progression of iDCM following chronic myocarditis. Methods: Patients were separated in inflammatory-positive and -negative groups according to expression of inflammatory markers (CD3, CD45Ro, and CD68). From a total of 32 iDCM patients (25 males and 7 females; mean age 43 6 12 years), serum and biopsy samples were investigated. Secreted TGF-b1, IL-6, TNF-a and IL-1b cytokines, the collagen synthesis/degradation (PICP/ICTP) marker, indicators of apoptosis (Caspases 3, 9, 8, Bax, Bcl2) and the marker of extracellular matrix degradation/inhibition (MMP9/TIMP-1) were assayed by ELISA. Fibrosis and inflammatory infiltrates were studied by immunohistochemistry. The statistical significance of data was analyzed by the Student t test (significance was set at p ! 0.05). Results: TGF-b1, a main regulator of collagen synthesis and fibrosis, was upregulated in serum and biopsy samples by 1.4 and 2.5 folds, respectively, but did not enhance myocardial fibrosis (being 1.1-1.2 folds lover than in controls). TGF-b1 significantly (p ! 0.05) correlated with upregulation of MMP9/TIMP1 in serums and biopsies, suggesting their involvement in TGF-b1 activation. Additionally, in biopsies TGF-b1 significantly (p ! 0.05) correlated with increase of pro-apoptotic markers, such as FasR, FasL, caspase-8, -3, and with depression of anti-apoptotic Bcl-2. Ratios of collagen synthesis and degradation (PICP/ICTP) in biopsies and sera were also downregulated from 59.6 to 10.34 and from 5.88 to 0.079, respectively. Parallelly, the amounts of secreted TGF-b1 significantly (p ! 0.05) correlated with caspase-8 in biopsies. Conclusion: We suggest that upregulation of TGF-b1 in biopsies and sera from iDCM patients is a consequence of low intensity inflammation with resulting activation of the extracellular matrix degradation complex MMP9/TIMP1 and collagen degradation. TGF-b1 upregulation, however, was not enough to activate collagen synthesis or to induce myocardial fibrosis. Rather such upregulation was found related to apoptosis but not to fibrosis. The intensity of inflammation in chronic myocarditis followed iDCM is too low to induce significant fibrogenesis.
205 PERSONALIZED MEDICINE: Prospective Patient Selection Utilizing an ADRB1 Genotype Assay in the GENETIC-AF Clinical Trial Melissa Barhoover1, Jeff Albrecht2, David Port3, Christopher Dufton4; 1LabCorp Clinical Trials, Durham, NC; 2LabCorp Clinical Trials, Los Angeles, CA; 3 University of Colorado Health Sciences Center, Aurora, CO; 4ARCA biopharma, Westminster, CO Introduction: In a previously reported pharmacogenomic sub-study of the BEST trial, the 1040 patient BEST DNA sub-study, patients with the b1389Arg/Arg genotype exhibited a higher clinical efficacy in response to treatment with the b-blocker/ sympatholytic agent bucindolol compared to patients with the b1389 Gly genotype. The results of the sub-study show a significant and substantial decrease in new-onset AF that was exclusively identified in patients with the b1389Arg/Arg genotype. A prospective patient selection study (GENETIC-AF) is underway to confirm these initial Results LabCorp has developed and validated a Clinical Trials Assay for ADRB1 Genotype to support ARCA biopharma’s GENETIC-AF clinical trial. Hypothesis: Utilizing a personalized medicine approach to only enroll patients with the b1389Arg/Arg genotype will allow for more efficient clinical trial design with a smaller patient population. This subset of patients may have a more efficacious treatment response to bucindolol, evaluated by the recurrence of symptomatic AF following electrical cardioversion in patients with chronic heart failure. The development of laboratory-approved tests as companion diagnostics is supported by Regulatory authorities as an alternative mechanism to the traditional IVD kit for personalized medicine assays. Methods: The LabCorp ADRB1 Genotype Assay is a TaqManÒ-based (Roche Molecular Systems) PCR assay used to determine the genotype of the b1 adrenergic receptor (ADRB1) at amino acid position 389 as either Arg or Gly using genomic DNA isolated from whole blood. The specific primers and probes for the LabCorp ADRB1 Genotype Assay were custom designed by LabCorp. The accuracy of the ADRB1 assay was established by direct comparison of results with those generated by the “gold standard” of DNA sequencing. Results: The LabCorp ADRB1 Genotype Assay was validated for use in selecting patients for the GENETIC-AF clinical trial for the following performance parameters: analytical accuracy, intra-assay precision, inter-assay precision, analytical specificity, analytical sensitivity and analyte stability. The results show 100% agreement between the
ADRB1 SNP assay results and the sequencing results, 100% concordance between replicates in a single run, between replicates tested over three days, and between the determined and expected results for the samples tested in the presence of potentially interfering substances. The assay sensitivity was determined to be less than 2.0 ng/mL, but greater than 0.5 ng/mL, and the samples were found to be stable up to one week at ambient temperature and an additional week at 4 C. Conclusions: The LabCorp ADBR1 Genotype Assay is analytically validated and fit for use in a clinical trial for patient selection. FDA has approved the IDE for the LabCorp ADBR1 Genotype Assay and the GENETIC-AF trial is currently on-going.
206 Increased Cholesterol Efflux Capacity is Associated with Improved Survival in Heart Transplant Recipients Ali Javaheri1, Payman Zamani1, Maria Molina1, Amrith Rodriguez1, Susan Chambers1, Patricia Stutman1, Wilhelmina Maslanek1, Mary Williams1, Daniya Lukmanova1, Antonino Picataggi1, Scott Lilly2, Jason Christie1, Y. Joseph Woo3, Lee R. Goldberg1, Jeffrey Billheimer1, Daniel J. Rader1; 1University of Pennsylvania, Philadelphia, PA; 2Ohio Sate University, Columbus, OH; 3Stanford University, Palo Alto, CA Objectives: To assess the relationship between pathways that impact cholesterol removal from cells and survival in cardiac transplant recipients. Introduction: In cardiac transplant recipients, high density lipoprotein (HDL) exhibits impaired ability to promote cholesterol efflux from macrophages, and coronary allograft vasculopathy (CAV) is a major cause of mortality. Given that cholesterol-loading of macrophages may play a role in CAV and that statins reduce macrophage foam cells and improve post-transplant survival, we sought to evaluate the relationship of capacity of HDL to promote removal of cholesterol from macrophages and mortality in patients post cardiac transplantation. Methods and Results: We used a case-cohort design to evaluate mortality risk associated with HDL cholesterol efflux capacity. From a cohort of cardiac transplant patients presenting to our echocardiography and cardiac catheterization laboratories, we enrolled cases who died between 2009-2012 (n534). Controls (n557) were identified as cardiac transplant patients who were alive on October 1st, 2013. Clinical characteristics, serum lipids, apolipoproteins, and cholesterol efflux capacity were compared between groups. Efflux capacity was measured by incubating apolipoprotein B-depleted serum with macrophages in a validated ex vivo system. Multivariable cox proportional hazard ratios demonstrated that chronic kidney disease, CAV, and smoking were associated with increased mortality, while higher levels of cholesterol efflux capacity of HDL (HR 0.63, 95% CI 0.40-0.995, p50.048 Table) were protective. An interaction effect was observed between efflux capacity and the serum levels of apolipoprotein E (p50.002). The relationship between HDL cholesterol efflux capacity and mortality persisted after correction for HDL cholesterol mass and was particularly notable amongst those patients with CAV (HR 0.35, 95% CI 0.18-0.70, p50.003). Conclusion: Higher HDL cholesterol efflux capacity is associated with improved survival after cardiac transplantation independent of HDL cholesterol levels. This finding suggests that interventions to increase HDL cholesterol efflux capacity may provide benefit in cardiac transplant recipients, in particular those with allograft vasculopathy. Table. Variables associated with mortality post-cardiac transplant adjusted for clinical variables
Variable
HR (95% CI)
Efflux quartile CAV Current smoking CKD (Stage III or IV)
0.63 1.92 3.16 4.73
(0.40-0.995) (0.92-3.99) (1.03-9.70) (1.61-13.83)
p-value 0.048 0.082 0.005 0.044
207 Epigenetic Regulation of Gene Expression by Long Intergenic Non-Coding RNA Encoded by Cardiac Myosin Heavy Chain Genes Prabhu Mathiyalagan, Jun Okabe, Harikrishnan Kaipananickal, Xiao-Jun Du, Assam El-Osta; BakerIDI Heart and Diabetes Institute, Melbourne, Australia Background: Cardiac pressure overload induced prototypical shift in myosin heavy chain (MHC) gene expression is regulated by complex interactions of MHC-encoded long noncoding RNA (lncRNA), primary microRNA-208b (pri-miR-208b) and long intergenic ncRNA (lincRNA), antisense (AS) b-MHC. The precise mechanism by which AS b-MHC acts as a mediator integrating epigenetic modifications to regulate gene expression in the mouse heart remains unknown. Objective: In this study, we explored the expression as well as gene-specific interaction of cardiac lincRNA, AS b-MHC in a mouse model to attenuate pressure overload caused by transverse aortic constriction (TAC) using the prototypical histone deacetylase (HDAC)
HFSA
S81
Molecular Biology and Genetics II 204 Mechanism of Fibrosis in Inflammatory Dilated Cardiomyopathy Daiva Bironaite1, Dainius Daunoravicius2, Julius Bogomolovas3, Sigitas Cibiras2, Dalius Vitkus2, Edvardas Zurauskas2, Ieva Zasytyte2, Siegfried Labeit3, Algirdas Venalis1, Virginija Grabauskiene2; 1State Research Institute, Center for Innovative Medicine, Vilnius, Lithuania; 2Vilnius University, Faculty of Medicine, Vilnius, Lithuania; 3Universit€atsmedizin Mannheim, Mannheim, Germany Background: Inflammation plays a significant role in myocardial damage and is considered to be involved in the pathogenesis of myocarditis, which often progresses to inflammatory dilated cardiomyopathy (iDCM). Myocarditis is known to result in fibrosis, inflammatory exudates, adverse ventricular remodeling and, eventually, cardiac failure. The level of fibrosis in iDCM is supposed to vary depending on inflammatory intensity and duration. Objectives: The purpose of our study was to investigate the molecular mechanisms regulating fibrogenesis and its role in progression of iDCM following chronic myocarditis. Methods: Patients were separated in inflammatory-positive and -negative groups according to expression of inflammatory markers (CD3, CD45Ro, and CD68). From a total of 32 iDCM patients (25 males and 7 females; mean age 43 6 12 years), serum and biopsy samples were investigated. Secreted TGF-b1, IL-6, TNF-a and IL-1b cytokines, the collagen synthesis/degradation (PICP/ICTP) marker, indicators of apoptosis (Caspases 3, 9, 8, Bax, Bcl2) and the marker of extracellular matrix degradation/inhibition (MMP9/TIMP-1) were assayed by ELISA. Fibrosis and inflammatory infiltrates were studied by immunohistochemistry. The statistical significance of data was analyzed by the Student t test (significance was set at p ! 0.05). Results: TGF-b1, a main regulator of collagen synthesis and fibrosis, was upregulated in serum and biopsy samples by 1.4 and 2.5 folds, respectively, but did not enhance myocardial fibrosis (being 1.1-1.2 folds lover than in controls). TGF-b1 significantly (p ! 0.05) correlated with upregulation of MMP9/TIMP1 in serums and biopsies, suggesting their involvement in TGF-b1 activation. Additionally, in biopsies TGF-b1 significantly (p ! 0.05) correlated with increase of pro-apoptotic markers, such as FasR, FasL, caspase-8, -3, and with depression of anti-apoptotic Bcl-2. Ratios of collagen synthesis and degradation (PICP/ICTP) in biopsies and sera were also downregulated from 59.6 to 10.34 and from 5.88 to 0.079, respectively. Parallelly, the amounts of secreted TGF-b1 significantly (p ! 0.05) correlated with caspase-8 in biopsies. Conclusion: We suggest that upregulation of TGF-b1 in biopsies and sera from iDCM patients is a consequence of low intensity inflammation with resulting activation of the extracellular matrix degradation complex MMP9/TIMP1 and collagen degradation. TGF-b1 upregulation, however, was not enough to activate collagen synthesis or to induce myocardial fibrosis. Rather such upregulation was found related to apoptosis but not to fibrosis. The intensity of inflammation in chronic myocarditis followed iDCM is too low to induce significant fibrogenesis.
205 PERSONALIZED MEDICINE: Prospective Patient Selection Utilizing an ADRB1 Genotype Assay in the GENETIC-AF Clinical Trial Melissa Barhoover1, Jeff Albrecht2, David Port3, Christopher Dufton4; 1LabCorp Clinical Trials, Durham, NC; 2LabCorp Clinical Trials, Los Angeles, CA; 3 University of Colorado Health Sciences Center, Aurora, CO; 4ARCA biopharma, Westminster, CO Introduction: In a previously reported pharmacogenomic sub-study of the BEST trial, the 1040 patient BEST DNA sub-study, patients with the b1389Arg/Arg genotype exhibited a higher clinical efficacy in response to treatment with the b-blocker/ sympatholytic agent bucindolol compared to patients with the b1389 Gly genotype. The results of the sub-study show a significant and substantial decrease in new-onset AF that was exclusively identified in patients with the b1389Arg/Arg genotype. A prospective patient selection study (GENETIC-AF) is underway to confirm these initial Results LabCorp has developed and validated a Clinical Trials Assay for ADRB1 Genotype to support ARCA biopharma’s GENETIC-AF clinical trial. Hypothesis: Utilizing a personalized medicine approach to only enroll patients with the b1389Arg/Arg genotype will allow for more efficient clinical trial design with a smaller patient population. This subset of patients may have a more efficacious treatment response to bucindolol, evaluated by the recurrence of symptomatic AF following electrical cardioversion in patients with chronic heart failure. The development of laboratory-approved tests as companion diagnostics is supported by Regulatory authorities as an alternative mechanism to the traditional IVD kit for personalized medicine assays. Methods: The LabCorp ADRB1 Genotype Assay is a TaqManÒ-based (Roche Molecular Systems) PCR assay used to determine the genotype of the b1 adrenergic receptor (ADRB1) at amino acid position 389 as either Arg or Gly using genomic DNA isolated from whole blood. The specific primers and probes for the LabCorp ADRB1 Genotype Assay were custom designed by LabCorp. The accuracy of the ADRB1 assay was established by direct comparison of results with those generated by the “gold standard” of DNA sequencing. Results: The LabCorp ADRB1 Genotype Assay was validated for use in selecting patients for the GENETIC-AF clinical trial for the following performance parameters: analytical accuracy, intra-assay precision, inter-assay precision, analytical specificity, analytical sensitivity and analyte stability. The results show 100% agreement between the
ADRB1 SNP assay results and the sequencing results, 100% concordance between replicates in a single run, between replicates tested over three days, and between the determined and expected results for the samples tested in the presence of potentially interfering substances. The assay sensitivity was determined to be less than 2.0 ng/mL, but greater than 0.5 ng/mL, and the samples were found to be stable up to one week at ambient temperature and an additional week at 4 C. Conclusions: The LabCorp ADBR1 Genotype Assay is analytically validated and fit for use in a clinical trial for patient selection. FDA has approved the IDE for the LabCorp ADBR1 Genotype Assay and the GENETIC-AF trial is currently on-going.
206 Increased Cholesterol Efflux Capacity is Associated with Improved Survival in Heart Transplant Recipients Ali Javaheri1, Payman Zamani1, Maria Molina1, Amrith Rodriguez1, Susan Chambers1, Patricia Stutman1, Wilhelmina Maslanek1, Mary Williams1, Daniya Lukmanova1, Antonino Picataggi1, Scott Lilly2, Jason Christie1, Y. Joseph Woo3, Lee R. Goldberg1, Jeffrey Billheimer1, Daniel J. Rader1; 1University of Pennsylvania, Philadelphia, PA; 2Ohio Sate University, Columbus, OH; 3Stanford University, Palo Alto, CA Objectives: To assess the relationship between pathways that impact cholesterol removal from cells and survival in cardiac transplant recipients. Introduction: In cardiac transplant recipients, high density lipoprotein (HDL) exhibits impaired ability to promote cholesterol efflux from macrophages, and coronary allograft vasculopathy (CAV) is a major cause of mortality. Given that cholesterol-loading of macrophages may play a role in CAV and that statins reduce macrophage foam cells and improve post-transplant survival, we sought to evaluate the relationship of capacity of HDL to promote removal of cholesterol from macrophages and mortality in patients post cardiac transplantation. Methods and Results: We used a case-cohort design to evaluate mortality risk associated with HDL cholesterol efflux capacity. From a cohort of cardiac transplant patients presenting to our echocardiography and cardiac catheterization laboratories, we enrolled cases who died between 2009-2012 (n534). Controls (n557) were identified as cardiac transplant patients who were alive on October 1st, 2013. Clinical characteristics, serum lipids, apolipoproteins, and cholesterol efflux capacity were compared between groups. Efflux capacity was measured by incubating apolipoprotein B-depleted serum with macrophages in a validated ex vivo system. Multivariable cox proportional hazard ratios demonstrated that chronic kidney disease, CAV, and smoking were associated with increased mortality, while higher levels of cholesterol efflux capacity of HDL (HR 0.63, 95% CI 0.40-0.995, p50.048 Table) were protective. An interaction effect was observed between efflux capacity and the serum levels of apolipoprotein E (p50.002). The relationship between HDL cholesterol efflux capacity and mortality persisted after correction for HDL cholesterol mass and was particularly notable amongst those patients with CAV (HR 0.35, 95% CI 0.18-0.70, p50.003). Conclusion: Higher HDL cholesterol efflux capacity is associated with improved survival after cardiac transplantation independent of HDL cholesterol levels. This finding suggests that interventions to increase HDL cholesterol efflux capacity may provide benefit in cardiac transplant recipients, in particular those with allograft vasculopathy. Table. Variables associated with mortality post-cardiac transplant adjusted for clinical variables
Variable
HR (95% CI)
Efflux quartile CAV Current smoking CKD (Stage III or IV)
0.63 1.92 3.16 4.73
(0.40-0.995) (0.92-3.99) (1.03-9.70) (1.61-13.83)
p-value 0.048 0.082 0.005 0.044
207 Epigenetic Regulation of Gene Expression by Long Intergenic Non-Coding RNA Encoded by Cardiac Myosin Heavy Chain Genes Prabhu Mathiyalagan, Jun Okabe, Harikrishnan Kaipananickal, Xiao-Jun Du, Assam El-Osta; BakerIDI Heart and Diabetes Institute, Melbourne, Australia Background: Cardiac pressure overload induced prototypical shift in myosin heavy chain (MHC) gene expression is regulated by complex interactions of MHC-encoded long noncoding RNA (lncRNA), primary microRNA-208b (pri-miR-208b) and long intergenic ncRNA (lincRNA), antisense (AS) b-MHC. The precise mechanism by which AS b-MHC acts as a mediator integrating epigenetic modifications to regulate gene expression in the mouse heart remains unknown. Objective: In this study, we explored the expression as well as gene-specific interaction of cardiac lincRNA, AS b-MHC in a mouse model to attenuate pressure overload caused by transverse aortic constriction (TAC) using the prototypical histone deacetylase (HDAC)